This invention relates to the lipase catalysed esterification of marine oils.
It is well known in the art to refine oil products of various kinds, including marine oils, with the aid of lipase catalysts whose specificity under the refining conditions employed enhances the recovery of a desired product.
For example, in PCT/WO95/00050 we disclosed a process for treating an oil composition containing saturated and unsaturated fatty acids in the form of triglycerides to transesterification reaction conditions with a C1-6 alcohol such as ethanol under substantially anhydrous conditions in the presence of a lipase active to preferentially catalyse the transesterification of the saturated and monounsaturated fatty acids. With the preferred lipases, Pseudomonas sp. lipase (PSL) and Pseudomonas fluorescens lipase (PFL) it was possible to prepare from marine oil sources concentrates containing more than 70% by weight of the commercially and therapeutically important omega-3 polyunsaturated fatty acids EPA (eicosapentaenoic acid, C20:5) and DHA (docosahexaenoic acid, C22:6) in the form of glycerides.
A number of lipase-catalysed refining processes have utilised glycerol.
By way of example, JP 62-91188 (1987) teaches a process for preparing glycerides of polyunsaturated fatty acids (PUFA) in which the PUFA as free acid or ester is reacted with glycerol in the presence of a thermostable lipase. The fatty acid composition of the resulting glyceride product is substantially the same as in the starting PUFA.
WO91/16443 discloses a process for converting PUFA into triglycerides. The free fatty acids, for example mixtures of EPA and DHA, are reacted with about stoichiometric amounts of glycerol in the presence of a lipase, especially Candida antarctica, under essentially anhydrous, organic solvent-free, elevated temperature conditions with continuous removal of water and volatile alcohols. We are aware that there was little or no discrimination between EPA and DHA in this process.
In a paper in Int. J. Food Sci. Technol. (1992), 2, 73-76, Lie and Molin describe the esterification of a fish oil fatty acid concentrate with glycerol using three different lipases, including MML. Under the conditions used (5% water) they obtained a DHA-depleted free acid fraction (about 50% of the starting material) and a glyceride fraction with the same EPA content as the original fish oil concentrate. Thus, some selectivity was observed.
A paper by Myrnes et al in JAOCS, Vol. 72, No. 11 (1995), 1339-1344 discloses an organic solvent-free, lipase-catalysed glycerolysis of marine oils. A variety of different lipases are tested, and the reactions are run at low temperatures (12xc2x0 C. or less) in the presence of relatively high (3.6%) amounts of water. Analysis of the resulting monoglyceride fraction showed, in some cases, good selectivity between unsaturated and saturated fatty acids, but no significant differences between individual PUFA.
Moore et al in JAOCS, Vol. 73, No. 11 (1996), 1409-1414 teach the hydrolysis of a fish oil in the presence of Candida rugosa lipase (CRL) to produce separate DHA-enriched and EPA-enriched fractions.
Subsequently, the EPA-enriched free fatty acid fraction is re-esterified with glycerol in the presence of Rhizomucor miehei lipase (MML).
A paper by McNeill et al in JAOCS, Vol. 73, No. 11 (1996), 1403-1407 discloses a MML-catalysed esterification of a n-3 PUFA concentrate with stoichiometric amounts of glycerol at 55xc2x0 C. with continuous removal of water. The resulting triglyceride fraction contained the same level of DHA as the feed.
Finally, mention is made of WO96/37586 and WO96/37587. Example 3 of WO96/37586 discloses a process in which a free fatty acid concentrate originating in Chilean Fish Oil, comprising (after solvent fractionations of sodium salts) 25% EPA and 18% DHA, was directly esterified with glycerol using an immobilized Candida rugosa lipase (CRL) in the presence of 10% water at 35xc2x0 C. After 120 hours, the extent of conversion had reached about 60%. In the glyceride mixture obtained, the triglycerides contained 28.2% EPA and 3.8% DHA and the monoglyceride fraction had 28.9% EPA and 4.5% DHA. The residual free fatty acids comprised 23.2% EPA and 31.5% DHA. This indicates good selectivity between EPA and DHA.
In contrast, in Examples 1 and 2, the MML catalysed re-esterification of a free fatty acid fraction with glycerol did not show significant selectivity between EPA and DHA.
The disclosure of WO96/37587 is similar to that of WO96/37586. Examples 1, 4, 6 and 8 show the glycerolysis of PUFA with MML without any discrimination between EPA and DHA.
It will be apparent from this, by no means exhaustive, discussion of the prior art that extensive research has been carried out in order to develop lipase-catalysed processes for isolating such commercially important PUFA as EPA and DHA from compositions such as fish oils containing them in relatively low concentrations.
We have now discovered a lipase-catalysed process for preparing concentrates of EPA and DHA by the direct esterification of free fatty acid from fish oil which, by selection of the lipase, permits the EPA/DHA contents of the resulting concentrate to be tailored to meet customers"" different requirements.
More particularly, the present invention provides a process for esterifying a marine oil composition containing EPA and DHA as free fatty acids to form a free fatty acid fraction enriched in at least one of these fatty acids as compared to the starting composition, comprising the step of reacting said marine oil composition with glycerol in the presence of a lipase catalyst under reduced pressure and essentially organic solvent-free conditions, and recovering a free fatty acid fraction enriched in at least one of EPA and DHA.
The present invention is predicated on the discovery that glycerol can act as an excellent substrate for a lipase-catalysed direct esterification of marine oil free fatty acids, provided that certain critical reaction conditions are followed. This finding was not at all to be expected in view of the prior research using glycerol referred to above. The main esterification reaction can be schematically represented by the following equation in which the lipase catalyst is Rhizomucor miehei (MML): 
The product also contains other types of EPA-enriched glycerides, not shown in the schematic equation.
As will be discussed in more detail below, and illustrated in Example 8, the selection of the lipase catalyst can crucially affect the nature of the product. In the case of MML used in the illustrated reaction scheme, the product is a DHA-enriched free fatty acid fraction and an EPA-enriched glyceride fraction.
A significant feature of the present process is that it takes advantage of the fact that the selectivity of a lipase towards individual fatty acids is greater when they are in the form of free acids rather than as glycerides, since complications related to lipase regioselectivity or positioned selectivity are avoided. Surprisingly, the reaction with glycerol is far less successful when the EPA and DHA are present as esters, rather than as free acids, as is shown in Example 10 (Comparative) below.
The use in accordance with the present invention of glycerol as the substrate has the further advantage that it aids separation of the glyceride and free fatty acid product fractions by molecular distillation. The reason for this is considered to be that the esters of a trioic alcohol such as glycerol are less volatile than similar esters of short-chain alcohols such as methanol, ethanol and propanol.
It has been found that the relative amounts of glycerol are important to make the esterification reaction succeed. Preferably, a molar ratio of glycerol to free fatty acids in the starting composition of from 1:1.5 to 1:3 should be used, more preferably from 1:1.5 to 1:2.5. In our experimental work to date we have found that a molar ratio of about 1:2 of glycerol to fatty acids is optimal (corresponding to a ratio of available hydroxyl groups to free fatty acids of 1.5:1).
It is essential that the esterification reaction should be earned out under reduced pressure, in order to remove water from the reaction system as it is formed. This is necessary in order to make the reaction non-reversible, thereby making it possible to obtain high recoveries of the desired EPA/DHA products, Thus, the esterfication will generally be carried out at a pressure below 6665 Pa, and normally below 1.333 Pa e.g. from 133.3-1.333 Pa, although we have made the surprising observation that the reduced pressure conditions for optimum lipase activity is dependent to some extent on the particular lease used. Thus. in some cases it may be advantageous to use a pressure of front 1.333 to 133.3 Pa, and in the Examples which follow we report excellent results with pressures as low as 1.333-13.33 Pa. The optimum low pressure conditions for the particular lipase being used can, of course, be readily determined by routine experiments.
Organic solvents should be absent from the present process, unlike many prior art lipase-based systems, because organic solvents are volatile and will evaporate off under vacuum conditions.
The temperature at which the esterification reaction is conducted will depend on the marine oil composition being treated as well as on the lipase being used. It is desirable that the viscosity of the marine oil composition should be sufficiently low to enable the composition to be adequately agitated during the reaction, and for this reason it is often necessary to use temperatures of at least 20xc2x0 C. On the other hand, too high temperatures are undesirable because high temperatures work against the kinetic resolution on which the fatty acid lipase discrimination is based, and also because the EPA and DHA can be destroyed by prolonged exposure to high temperatures, while lipases are also intolerant of high temperatures. Bearing factors such as these in mind, it is generally preferred to operate within the range of 20-40xc2x0 C., often most preferably at 37-40xc2x0 C., although temperatures of 0-20xc2x0 C. may be used for fish oil compositions of high EPA and/or DHA contents where the composition remains sufficiently liquid at these low temperatures and conversely higher temperatures, in the range 40-70xc2x0 C. may be possible for such stable immobilized lipases as MML and CAL.
The starting material for the present process can be any composition from a marine source containing EPA and DHA in free acid form. Such a composition may be obtained by saponification of crude fish oils, eg with sodium hydroxide, followed by acidification with eg sulphuric acid, according to standard procedures well known to those in the fish oil processing industries. Typically, the compositions will contain total contents of EPA and DHA in free acid form of 15-35% by weight, preferably 25-35%. Fish oils which are rich in DHA, such as tuna oil containing about 5% EPA and 25% DHA by weight are particularly suitable for preparing DHA concentrates by the process of the present invention, whilst fish oils rich in EPA (e.g. sardine oil with about 18% EPA and 12% DHA by weight) and Chile fish oil (20% EPA and 7% DHA by weight) are especially suitable raw materials for making EPA concentrates. However, it is an advantage of the present invention that cheaper fish oils with lower total EPA and DHA contents such as herring oil (about 6% EPA and about 8% DHA by weight) can be used as starting materials for the preparation of EPA and/or DHA-enriched fractions by the process of this invention, as shown in the Examples which follow later in this specification.
As mentioned earlier in this specification, it is a feature of the present process that it is possible to vary the nature of the enriched fractions by the choice of the lipase used. For example, the following effects are observed with the lipases noted:
i. a DHA-enriched free fatty acid fraction and an EPA-enriched glyceride fraction is obtained with with Rhizomucor meihei lipase (MML), Mucor javanicus lipase (MJL), and Aspergillus niger lipase (ANL); and
ii. an EPA/DHA-endriched free fatty acid fraction and a glyceride fraction enriched in saturated fatty acids is obtained with Pseudomonas sp.xe2x80x94Amano AK (PSL), Pseudomonas fluorescensxe2x80x94Amano PS (PFL), Rhizopus oryzaexe2x80x94Amano F (ROL) and Humicula Lanuginosaxe2x80x94Amano CE (HLL),
This ability to vary the nature of the product by appropriate selection of the lipase catalyst has the advantage that the operaton of the process can be tailored to suit customers"" particular requirements. For example, one customer may require a DHA concentrate for supplementing infant feed, while another customer may require a mixed EPA/DHA concentrate for manufacturing a health product, but the requirements of both customers can be met simply by changing the lipase catalysts used.
Of course, yet more possibilities for tailoring the composition of the final product may be had by carrying out the process in two or more separate stages, with different lipase catalysts being used in the different stages.
The preferred lipases for the present process are Rhizomucor miehei (MML), which discriminates strongly between EPA and DHA; and Pseudomonas sp. (PSL), which discriminates between EPA and DHA, on the one hand, and the remaining fatty acids in fish oil on the other.
It is preferred, at least on the industrial scale, to use an immobilized form of the selected lipase, since it is found that not only does immobilization often increase the activity of the enzyme, especially at very low pressures, of the order of 1.333 to 133.3 Pa, but it also improves its stability and aids its recovery, which are all factors which affect the economics of the process.
Sufficient of the lipase should be used in order to effect the desired esterification reaction. In our work with immobilized MML we have used about 10% by weight of the immobilized product, based on the content of fatty acids in the marine composition being treated, which corresponds to a concentration of MML of about 1% by weight (the commercially available immobilized MML being about 10% lipase and 90% carrier).
In contrast, using non-immobilized lipases, we have utilized lipase concentrations of 10% by weight of the fatty acid content.
Following the completion of the esterification reaction, the product is separated in fractions containing mainly free fatty acids and glycerides respectively, by, molecular distillation.
The molecular distillation step to separate the free fatty acid fraction from the glyceride fraction can be performed at a temperature ranging from 100-200xc2x0 C., but will normally lie in the range of 140-180xc2x0 C. Its successfulness in terms of the achievable ratio residium/destillate will depend on the vacuum. The vacuum may vary depending on factors such as the volatile components present in the mixture. It will generally be in the range of 1xc3x9710xe2x88x924-1xc3x9710xe2x88x922 mbar, but a person skilled in the art can use the combination of the achievable vacuum, which in some instances may be outside the mentioned range, and a suitable temperature to achieve the desired end result.
Of course, the product from a first lipase-catalysed esterification may then be further concentrated in one or more subsequent lipase-catalysed esterifications, using the same or different lipase.
The free acid fraction which is obtained at the conclusion of the process may either be used as such, or if a product in free acid form is not acceptable for the intended use, then it can first be converted into ethyl ester, glyceride or other more acceptable form by any suitable method.
Likewise, in the case where the separated glyceride fraction contains EPA or DHA in economically worthwhile concentrations, this fraction may also be subjected to further treatment, for instance hydrolysis with aqueous alkali to form free acids, or esterification with ethanol to form ethyl esters of the fatty acids. The free fatty acid or ethyl ester fraction, thus formed may then, if desired, be further concentrated, e.g. by molecular distillation.
The esterification process of the present invention has a number of advantages which render it particularly suitable for industrialisation. The ability to tailor the composition of the products, especially by selection of the lipase catalyst, has already been mentioned, but further advantages which make the process attractive commercially include:
i. the high yields of highly concentrated EPA, DHA or EPA+DHA products which can be made,
ii. the absence of any organic solvents, thus not only obviating the purification problems which the presence of such solvents can often cause, but also reducing the bulkiness of the process, which is important economically (less energy requirements, etc),
iii. the ability to re-use immobilized lipase catalysts in several, perhaps up to 20 or more, successive runs, thus again contributing to keep costs down,
iv. the ability to use any suitable marine oil composition which contains the polyunsaturated fatty acids of interest, and
v. the overall simplicity of the esterification and subsequent separation processes.